The isolation and purification of nucleic acids (DNA and RNA, for example) from complex matrices such as blood, bacterial cell culture media, and forensic samples is an important process in genetic research, nucleic acid probe diagnostics, forensic DNA testing and other areas. The separation of single-stranded from double-stranded DNA, and of bound from unbound nucleic acid hybridization probes are also important techniques in these areas. A variety of methods for preparing nucleic acids are known in the art; however, each has its limitations.
Traditionally a phenol chloroform extraction has been used, but this requires the use of toxic and corrosive chemicals and is not easily automated. Solid phase extraction has also been used for nucleic acid purification. For example, Boom et al. (U.S. Pat. No. 5,234,809) describe a method for isolating nucleic acids from a nucleic acid source in which a suspension of silica particles is mixed with a buffered chaotropic agent such as guanidinium thiocyanate in a reaction vessel followed by addition of the sample and thorough mixing. In the presence of the chaotrope, the nucleic acids are adsorbed onto the silica, which is separated from the liquid phase by centrifugation, washed with an alcohol water mix, and finally eluted using a dilute aqueous buffer. Silica solid phase extraction requires the use of the alcohol wash step to remove residual chaotrope without eluting the nucleic acid; however, great care must be taken to remove all traces of the alcohol (by heat evaporation or washing with another very volatile and flammable solvent) in order to prevent inhibition of sensitive enzymes used to amplify or modify the nucleic acid in subsequent steps.
Ion exchange methods, such as those offered by Qiagen (Valencia, Calif. 91355), produce high quality nucleic acids. However, these result in the presence of high levels of salts which must be removed before the nucleic acids can be further utilized.
The present invention provides methods for the isolation, including concentration, and preferably purification and recovery of nucleic acids. It also provides methods for the reduction in the amount of nucleic acid that adheres to a surface. In one embodiment, the method involves adhering nucleic acid to a hydrophobic organic polymeric material, such as polypropylene powder and polytetrafluoroethylene fibrils, and removing (e.g., eluting) the nucleic acids from such hydrophobic materials with a nonionic surfactant. In another embodiment, a nonionic surfactant is used to treat a hydrophobic surface to reduce, and preferably prevent, the adhesion of nucleic acids to hydrophobic surfaces.
Nucleic acids isolated according to the invention, will be useful, for example, in assays for detection of the presence of a particular nucleic acid in a sample. Such assays are important in the prediction and diagnosis of disease, forensic medicine, epidemiology, and public health. For example, isolated DNA may be subjected to hybridization and/or amplification to detect the presence of an infectious virus or a mutant gene in an individual, allowing determination of the probability that the individual will suffer from a disease of infectious or genetic origin. The ability to detect an infectious virus or a mutation in one sample among the hundreds or thousands of samples being screened takes on substantial importance in the early diagnosis or epidemiology of an at-risk population for disease, e.g., the early detection of HIV infection, cancer or susceptibility to cancer, or in the screening of newborns for diseases, where early detection may be instrumental in diagnosis and treatment. In addition, the method can also be used in basic research laboratories to isolate nucleic acid from cultured cells or biochemical reactions. The nucleic acid can be used for enzymatic modification such as restriction enzyme digestion, sequencing, and amplification.
In one preferred embodiment, a method for isolating nucleic acid from a sample includes: introducing a sample comprising target nucleic acid (e.g., DNA, RNA, PNA) to a hydrophobic organic polymeric solid phase to adhere at least a portion of the target nucleic acid to the solid phase; and applying a nonionic surfactant to the solid phase to remove at least a portion of the adhered target nucleic acid. Preferably, the sample is a biological sample, which include, for example cells. In certain embodiments, prior to introducing the biological sample, the method includes lysing the cells to release the contents of the cells as a lysate that includes nucleic acid.
In certain embodiments, the method further includes introducing a binding buffer comprising an added salt to the hydrophobic organic polymeric solid phase to assist in adhering the nucleic acid to the solid phase. Preferably, the binding buffer is introduced prior to introducing the sample. In certain embodiments, the method further includes washing the solid phase having nucleic acid adhered thereto to remove non-nucleic acid components of the sample. Such washing is typically with a washing buffer, comprising an added salt.
The hydrophobic organic polymeric solid phase material preferably includes a fluorinated polymer such as polytetrafluoroethylene. More preferably it includes a polyolefin such as polyethylene or polypropylene. The nonionic surfactant is preferably a polyoxyethylene surfactant and more preferably a polyoxyethylene-co-oxypropylene surfactant.
The present invention also provides a method for isolating double-stranded DNA from a sample. The method includes: introducing a sample comprising double-stranded DNA to a hydrophobic organic polymeric solid phase to adhere at least a portion of the double stranded DNA to the solid phase; washing the solid phase having double-stranded DNA adhered thereto to remove non-double-stranded DNA components of the sample (including single-stranded DNA); and applying a nonionic surfactant to the solid phase to remove at least a portion of the adhered double-stranded DNA. Thus, the methods of the present invention include the separation of various types of nucleic acid.
The present invention also provides a method for reducing the amount of (and preferably preventing) nucleic acid that adheres to a hydrophobic organic polymeric surface. The method includes applying a nonionic surfactant to the hydrophobic organic polymeric surface, washing the surface with a solvent (such as water or other solvent, such as that in which the surfactant is dissolved), and contacting the surface with a sample comprising the nucleic acid.
The present invention also provides a kit that includes a hydrophobic organic polymeric solid phase to which nucleic acid will adhere and a nonionic surfactant capable of removing at least a portion of the nucleic acid from the solid phase. Preferably, the kit further includes a flow-through receptacle.
Definitions
xe2x80x9cNucleic acidxe2x80x9d shall have the meaning known in the art and refers to both DNA and RNA, in a wide variety of forms, including, without limitation, double-stranded or single-stranded configurations, circular form, plasmids, relatively short oligonucleotides, peptide nulceic acids also called PNA""s (as described in Nielsen et al., Chem. Soc. Rev., 26, 73-78 (1997)), and the like.
xe2x80x9cIsolatedxe2x80x9d refers to nucleic acid that has been removed from the sample in which it is originally found. This includes simply concentrating the desired nucleic acid without necessarily removing any other materials other than the original solvent in the original sample. It also includes separating desired nucleic acid from other materials, e.g., cellular components such as proteins, lipids, salts, etc. More preferably, the isolated nucleic acid is substantially purified. xe2x80x9cSubstantially purifiedxe2x80x9d refers to nucleic acid that is at least 50%, preferably at least 80%, and more preferably at least 95%, pure with respect to removal of a contaminant, e.g., cellular components such as protein, lipid, or salt. These percentages refer to the amount of target nucleic acid (e.g., DNA, RNA, PNA) relative to the total amount of the target nucleic acid plus other (non-target) nucleic acid and contaminants, e.g., cellular components such as proteins, lipids, salts, etc., other than the solvent in the sample. Thus, the term xe2x80x9csubstantially purifiedxe2x80x9d generally refers to separation of a majority of cellular components or reaction contaminants from the sample, so that compounds capable of interfering with the subsequent use of the isolated nucleic acid are removed.
xe2x80x9cAdheres toxe2x80x9d or xe2x80x9cadherancexe2x80x9d or xe2x80x9cbindingxe2x80x9d refers to reversible binding via a wide variety of mechanisms, including weak forces such as Van der Waals interactions, electrostatic interactions, affinity binding, or physical trapping. The use of this term does not imply a mechanism of action, and includes adsorptive and absorptive mechanisms.
xe2x80x9cHydrophobic organic polymeric solid phasexe2x80x9d refers to a polymer made of repeating units, which may be the same or different, of organic compounds of natural and/or synthetic origin. This includes homopolymers and heteropolymers (e.g., copolymers, terpolymers, tetrapolymers, etc., which may be random or block, for example). A hydrophobic polymer has a critical surface tension of less than the surface tension of water (e.g., less than about 72 dynes/cm), and preferably less than the critical surface tension of nylon (e.g., less than about 43 dynes/cm). This term includes fibrous or particulate forms of a polymer, which can be readily prepared by methods well-known in the art. Such materials typically form a porous matrix, although for certain embodiments, the solid phase also refers to a solid surface, such as a nonporous sheet of organic polymeric material.
xe2x80x9cSurfactantxe2x80x9d refers to a substance that lowers the surface or interfacial tension of the medium in which it is dissolved. xe2x80x9cNonionic surfactantxe2x80x9d refers to a surfactant molecule whose polar group is not electrically charged.
It has been found that nucleic acids will adhere to hydrophobic organic polymeric materials, such as polypropylene powder and polytetrafluoroethylene fibrils, and that nucleic acids can be effectively removed from such hydrophobic materials with a nonionic surfactant. Furthermore, a nonionic surfactant can be used to treat a hydrophobic organic polymeric surface, such as a plastic sheet, to reduce, and preferably prevent, the adhesion of nucleic acids to hydrophobic surfaces.
The methods of the present invention can be used to isolate nucleic acids from a wide variety of samples, particularly biological samples such as body fluids (e.g., whole blood, blood serum, urine, saliva), various tissues, cell cultures, etc. The type of sample is not a limitation of the present invention. Biological samples are those of biological or biochemical origin. Those suitable for use in the methods of the present invention can be derived from mammalian, plant, bacterial, or yeast sources. The biological sample can be in the form of single cells or in the form of a tissue. Cells or tissue can be derived from in vitro culture.
For samples containing cells, the cell membranes are initially lysed to release the contents of the cells as a lysate containing nucleic acid. Lysis herein is the physical disruption of the membranes of the cells, referring to the outer cell membrane and, when present, the nuclear membrane. This can be done using standard techniques, such as by boiling, by treatment with chaotropic agents, by physical disruption or freeze/thawing. Typically, to isolate nucleic acids from a cell lysate, it is preferable to remove the proteins first. This can be done, for example, by treating with azlactone beads (e.g., those of the type commercially available as EMPHAZE AB1 beads from 3M Company, St. Paul, Minn. or those of the type disclosed in International Publication No. WO 94/00464 (3M Company)) or passing the sample through a cation exchange membrane or column at a pH sufficiently low to maintain the positive charge of the proteins.
The nucleic acids may be isolated (e.g., concentrated or separated from contaminants) according to the invention from an impure, partially pure, or a pure sample. The purity of the original sample is not critical, as nucleic acid may be isolated from even grossly impure samples. For example, nucleic acid may be removed from an impure sample of a biological fluid such as blood, saliva, or tissue. If an origincal sample of higher purity is desired, the sample may be treated according to any conventional means known to those of skill in the art prior to undergoing isolation according to the invention. For example, the sample may be processed so as to remove certain impurities such as insoluble materials prior to nucleic acid isolation.
The nucleic acid isolated as described herein may be of any molecular weight and in single-stranded form, double-stranded form, circular, plasmid, etc. Various types of nucleic acid can be separated from each other (e.g., RNA from DNA, or double-stranded DNA from single-stranded DNA). For example, small oligonucleotides or nucleic acid molecules of about 10 to about 50 bases in length, much longer molecules of about 1000 bases to about 10,000 bases in length, and even high molecular weight nucleic acids of about 50 kb to about 500 kb can be isolated using the methods of the present invention. In some aspects, a nucleic acid isolated according to the invention may preferably be in the range of about 10 bases to about 100 kilobases.
The nucleic acid sample applied to the hydrophobic organic solid phase material according to the methods described herein may be in a wide variety of volumes. For example, the applied volume may be as large as 1 liter or as small as 1 xcexcL. It could also be utilized in a microfluidic format (the so called xe2x80x9clab on a chipxe2x80x9d) in which very small volumes (less than 1 xcexcL) are used. The nucleic acid applied to the solid phase matrix, as described herein, may be any amount, that amount being determined by the amount of solid phase. Preferably, the amount of nucleic acid applied to the solid phase is less than the dried weight of the solid phase, typically about {fraction (1/10,000)} to about {fraction (1/100)} (weight nucleic acid/solid phase). The amount of nucleic acid applied to the solid phase may be as much as 100 grams or as little as 1 picogram, for example.
The desired nucleic acid isolated from the solid phase material is preferably in an amount of about 30%, more preferably, about 70%, and most preferably, about 90%, or more than the amount of desired nucleic acid originally applied to the solid phase. Thus, the methods of the present invention provide for high recovery of the desired or target nucleic acid from a sample. Furthermore, exceedingly small amounts of nucleic acid molecules may be quantitatively recovered according to the invention. The recovery or yield is mainly dependent on the quality of the sample rather than the procedure itself. Because the invention provides a nucleic acid preparation that does not require concentration from a large volume, the invention avoids risk of loss of the nucleic acid.
Typically, a sample containing nucleic acid is added to the hydrophobic organic polymeric solid phase material in a flow-through receptacle, although this receptacle is not a necessary requirement. The nucleic acid then adheres to the hydrophobic organic polymeric solid phase material. Preferably, the binding of the nucleic acid may be enhanced by the use of a binding buffer, which can be added with or prior to the sample containing the target nucleic acid. Such a buffer typically includes a standard biological buffer that is compatible with nucleic acid and has a pH of about 3 to 11. Examples include AMPSO (3-[(1,1dimethyl-2-hydroxyethyl)amino]-2-hydroxypropanesulfonic acid (#A7585, Sigma Chemical Co, St. Louis, Mo. 63178), MES (2-[N-morpholino]ethansulfonic acid) (#M5287, Sigma Chemical Co, St. Louis, Mo. 63178), or PBS (phosphate buffered saline) (typically at about 20 Millimolar (mM) salt concentration). The binding buffer also typically includes added salt to promote hydrophobic binding. Examples include sodium salts of phosphate, perchlorate, citrate, or sulfate, at concentrations up to about 400 mM or even 1 molar, for example. Preferably, unbound materials, such as digested proteins, lipids, and other unwanted cellular components are then separated from the adhered nucleic acid by washing the nucleic acid/solid phase material with a buffer, for example. This washing buffer is typically a biological buffer that is compatible with the nucleic acid and typically within a pH range of about 3 to about 11. Examples of suitable washing buffers for removing undesirable materials include those listed above for the binding buffer. Such a buffer may or may not include added salts, such as those listed above for the binding buffer. Once these materials are removed, the nucleic acid may be recovered by removing (as by eluting) them from the solid phase material with a nonionic surfactant, which may be in an eluting buffer, e.g., such as those listed above (without added salt). Using this preferred method, the recovered nucleic acid is substantially pure, concentrated, and suitable for immediate use in subsequent experiments (e.g., sequencing experiments).
The hydrophobic organic polymeric solid phase material useful in the methods of the present invention may be a wide variety of organic materials that reversibly bind nucleic acid. Examples of suitable polymers include for example, polyolefins and fluorinated polymers. The solid phase material is typically washed to remove salts and other contaminants prior to use. It can either be stored dry or in aqueous suspension ready for use. The solid phase material is preferably used in a flow-through receptacle, for example, such as a pipet, syringe, or larger column, microtiter plate, or microfluidic device, although suspension methods that do not involve such receptacles could also be used.
The hydrophobic organic polymeric solid phase material useful in the methods of the present invention can include a wide variety of materials in a wide variety of forms. For example, it can be in the form of particles, which may be loose or immobilized, fibers, a microporous film, or membrane. For flow-through applications of the present invention, such materials are typically in the form of a porous matrix. Preferably, for such applications, the solid phase material has a relatively high surface area, such as, for example, more than one meter squared per gram (m2/g). For applications that do not involve the use of a flow-through device, such as pretreatment of a solid surface to prevent adhesion of nucleic acid, the solid phase material may or may not be in porous matrix.
In one embodiment, the solid phase material includes a fibril matrix, which may or may not have particles enmeshed therein. If both a fibril matrix and particles are used, at least one of them is hydrophobic and capable of binding the desired (i.e., target) nucleic acid. The fibril matrix can include any of a wide variety of fibers. Typically, the fibers are insoluble in an aqueous environment. Examples include glass fibers, polyolefin fibers, particularly polypropylene and polyethylene microfibers, aramid fibers, a fluorinated polymer, particularly, polytetrafluoroethylene fibers, and natural cellulosic fibers. Mixtures of fibers can be used, which may be active or inactive toward binding of nucleic acid. Preferably, the fibril matrix forms a web that is at least about 15 microns, and no greater than about 1 millimeter, and more preferably, no greater than about 500 microns thick.
If used, the particles are typically insoluble in an aqueous environment. They can be made of one material or a combination of materials, such as in a coated particle. They can be swellable or nonswellable, although they are preferably nonswellable in water and organic liquids. Preferably, if the particle is doing the adhering, it is made of nonswelling, hydrophobic material. They can be chosen for their affinity for the target nucleic acid. Examples of some water swellable particles are described in U.S. Pat. No. 4,565,663 (Errede et al.), U.S. Pat. No. 4,460,642 (Errede et al.), and U.S. Pat. No. 4,373,519 (Errede et al.). Particles that are nonswellable in water are described in U.S. Pat. No. 4,810,381 (Hagen et al.), U.S. Pat. No. 4,906,378 (Hagen et al.), U.S. Pat. No. 4,971,736 (Hagen et al.); and U.S. Pat. No. 5,279,742 (Markell et al.). Preferred particles are polyolefin particles, such as polypropylene particles (e.g., powder). Mixtures of particles can be used, which may be active or inactive toward binding of nucleic acid.
If coated particles are used, the coating is preferably an aqueous- or organic-insoluble, nonswellable material. The coating may or may not be one to which nucleic acid will adhere. Thus, the base particle that is coated can be inorganic or organic. The base particles can include inorganic oxides such as silica, alumina, titania, zirconia, etc., to which are covalently bonded organic groups. For example, covalently bonded organic groups such as aliphatic groups of varying chain length (C2, C4, C8, or C18 groups) can be used.
Examples of suitable solid phase materials that include a fibril matrix are described in U.S. Pat. No. 5,279,742 (Markell et al.), U.S. Pat. No. 4,906,378 (Hagen et al.), U.S. Pat. No. 4,153,661 (Ree et al.), U.S. Pat. No. 5,071,610 (Hagen et al.), U.S. Pat. No. 5,147,539 (Hagen et al.), U.S. Pat. No. 5,207,915 (Hagen et al.), and U.S. Pat. No. 5,238,621 (Hagen et al.). Those that include a polytetrafluoroethylene matrix (PTFE) are particularly preferred.
The PTFE matrix can be prepared according to the procedure described in U.S. Pat. No. 4,906,378 (Hagen et al.). Briefly, this involves the steps of blending the particulate material with a polytetrafluoroethylene aqueous dispersion in the presence of sufficient lubricant water to exceed the absorptive capacity of the solids, yet maintain a putty-like consistency, subjecting the putty-like mass to intensive mixing at a temperature of about 50xc2x0 C. to about 100xc2x0 C. to cause initial fibrillation of the polytetrafluoroethylene particles, biaxially calendering the putty-like mass to cause additional fibrillation of the polytetrafluoroethylene particles while maintaining the same water content, and drying the resultant sheet.
In another preferred embodiment, the solid phase (e.g., a microporous thermoplastic polymeric support) has a microporous structure characterized by a multiplicity of spaced, randomly dispersed, nonuniform shaped, equiaxed particles of thermoplastic polymer connected by fibrils. Particles are spaced from one another to provide a network of micropores therebetween. Particles are connected to each other by fibrils, which radiate from each particle to the adjacent particles. Either, or both, the particles or fibrils may be hydrophobic and allow for adherance of nucleic acids. Examples of preferred such materials have a high surface area, often as high as 40 meters2/gram as measured by Hg surface area techniques and pore sizes up to about 5 microns.
This type of fibrous material can be made by a preferred technique that involves involves the use of induced phase separation. This involves melt blending a thermoplastic polymer with an immiscible liquid at a temperature sufficient to form a homogeneous mixture, forming an article from the solution into the desired shape, cooling the shaped article so as to induce phase separation of the liquid and the polymer, and to ultimately solidify the polymer and remove a substantial portion of the liquid leaving a microporous polymer matrix. This method and the preferred materials are described in detail in U.S. Pat. No. 4,726,989 (Mrozinski), U.S. Pat. No. 4,957,943 (McAllister et al.), and U.S. Pat. No. 4,539,256 (Shipman). Such materials are referred to as thermally induced phase separation membranes (TIPS membranes) and are particularly preferred.
The affinity of the nucleic acids for the hydrophobic solid phase polymer can be controlled by the concentration of salt used during the binding or adhering of nucleic acids to the solid phase (which can be controlled through the use of a buffer referred to herein as a binding buffer), or the concentration of salt used during the elution step (which can be controlled through the use of a buffer referred to herein as an elution buffer). If desired, the binding and elution buffers, which typically are within a pH range of about 3 to about 11, can be used to separate different forms of nucleic acids. Depending on the solid phase, the binding of different types of nucleic acids can be controlled by the nature of the binding and elution buffers. High concentrations of salts, such as phosphates, will generally increase the binding capacity of solid supports such as polypropylene particulate. In some cases this can be used to separate different forms of nucleic acids.
A preferred aqueous solution for releasing the nucleic acids from the solid phase to form a solution containing nucleic acids includes a sufficient concentration of one or more nonionic surfactants. A preferred solution for releasing the nucleic acid includes water and a minimal amount of a buffer, such as PBS, to maintain the required pH and a minimal amount of nonionic surfactant. This minimal amount will depend upon which nonionic surfactant is used. For PLURONIC F68, this amount is no less than about 0.06 mM. In general, the concentration of nonionic surfactant needed for efficient elution must be higher than the critical micelle concentration (i.e., the minimum concentration of surfactant in water required to make a micelle, e.g., a submicroscopic aggregation of surfactant molecules) for that surfactant. Elution flow rate is not critical as long as there is enough time for the nucleic acid to desorb from the solid support.
A wide variety of suitable nonionic surfactants are known. They include, for example, polyoxyethylene surfactants, carboxylic ester surfactants, carboxylic amide surfactants, etc. Commercially available nonionic surfactants include, n-dodecanoylsucrose, n-dodecyl-xcex2-D-glucopyranoside, n-octyl-xcex2-D-maltopyranoside, n-octyl-xcex2-D-thioglucopyranoside, n-decanoylsucrose, n-decyl-xcex2-D-maltopyranoside, n-decyl-xcex2-D-thiomaltoside, n-heptyl-xcex2-D-glucopyranoside, n-heptyl-xcex2-D-thioglucopyranoside, n-hexyl-xcex2-D-glucopyranoside, n-nonyl-xcex2-D-glucopyranoside, n-octanoylsucrose, n-octyl-xcex2-D-glucopyranoside, cyclohexyl-n-hexyl-xcex2-D-maltoside, cyclohexyl-n-methyl-xcex2-D-maltoside, digitonin, and those available under the trade designations PLURONIC, TRITON, TWEEN, as well as numerous others commercially available and listed in the Kirk Othme Technical Encyclopedia. Preferred surfactants are the polyoxyethylene surfactants. More preferred surfactants are the polyoxyethylene co-oxypropylene surfactants.
According to a preferred embodiment of the present invention, a step is provided for recovering nucleic acids in a substantially purified form. This embodiment contemplates recovery methods that involve removal of either all of the eluate or aliquots of the eluate solution containing substantially purified nucleic acid released from the solid phase material. Such aliquots can be subjected to a variety of analytical and synthetic techniques known to one of skill in the art.
Examples of devices for using the methods of the present invention include standard laboratory filter holders and filters furnished by companies such as Millipore, Inc. (Bedford, Mass. 01730), Bio-Rad, Inc. (Hercules, Calif. 94547), Osmonics, Inc. (Westborough, Mass. 01581), and Whatman, Inc. (Clifton, N.J. 07014). The method of the invention can be conducted in filtration devices which facilitate the movement of solutions through filters (referred to as flow-through devices) by means including centrifugation, suction, pressure. Other devices include microtiter plates and microfluidic devices.
The present invention also provides a kit, which includes a solid support either with or without a holder (for example, a filter holder such as a syringe filter holder or a spin filter holder, or a column with retaining frits at each end for retaining particulate material), a nonionic surfactant, either neat or in a solution, and instructions for binding and eluting the nucleic acid. Preferably, the present invention provides kits that include a flow-through receptacle having a hydrophobic solid phase organic polymeric material therein and a nonionic surfactant.
The present invention also provides a method for reducing or preventing adhesion of nucleic acid to a hydrophobic organic material. However, unlike JP 2268682 (Hitachi Ltd.), which includes a nonionic surfactant in a mixture with the nucleic acid, the method of the present invention involves pretreating the material with a surfactant, preferably washing the material, and then contacting the material with the nucleic acid.